1. Field of the Invention
The present invention relates to a method of purifying thuringiensin, and more particularly to a method of purifying thuringiensin by using calcium silicate adsorption and dibasic sodium phosphate dissociation processes.
2. Description of the Related Arts
Traditional chemical insecticides are extensively applied in agriculture. However, their toxicity is also harmful to human and livestock, and the dosage used is increasing due to the increasing resistance of the insects to the chemical insecticides. Thus, the use of traditional chemical insecticides not only endangers agricultural workers, but also jeopardizes the health of consumers due to the residual pesticides on crops. Further, the impact of traditional chemical pesticides on the environment is of serious concern. Therefore, an insecticide obtained from nature and possessing high safety and low resistance is a key developing point in the field of insecticides.
Bacillus thuringiensis is a naturally occurring, soil borne organism that has gained a great deal of attention for its ability to express compounds which control certain insect pests. Thuringiensin, one of eight toxins produced by B. thuringiensis, is a metabolic product and a heat-stable .beta.-exotoxin, especially effective for fly control and often referred to as "fly factor". Since the discovery of thuringiensin (McConnell, E. and Richards, A. G., (1959) Can. J. Microbiol. 5:161-168), many aspects of its physical and biochemical properties, modes of action, and insecticidal/acaricidal selectivity have been described (Bond, R. P. M., et al. (1969) Biochem. J. 114:477-488). Thuringiensin (C.sub.22 H.sub.32 N.sub.5 O.sub.19.H.sub.2 O) is a heat-stable compound with a molecular weight of 701 daltons. Its chemical structure is similar to that of nucleotides. The mechanism of insecticidal action is through inhibition of the production of DNA-dependent RNA polymerase by competition with ATP (Lecadet, M. M. and De Barjac, H., in Davidson, E. W. (Ed.): Pathogenesis of invertebrate microbial disease, pp.293-321, Totowa, N.J., Allanheld and Osmun, 1981). This toxic action generally applies to orders of insects such as Coleoptera, Diptera, Hymenoptera, Isoptera, Lepidoptera, Orthoptera, Neuroptera, Hemiptera and Acari in the families Tetranychidae and Phytoseiidae (Bond, R. P. M., et al. in Burges, H. D. and Hussey, N. W. (Eds.): Microbial control of insects and mites, pp.275-303, London Academic Press, 1971; Hall, I. M., et al. (1971) J. Invertebr. Pathol. 18:359-362; Herbert, D. A. and Harper, J. D., (1986) J. Economic Entomol. 79:592-595; Hoy, M. A. and Ouyang, Y. L., (1987) J. Economic Entomol. 80:507-511). The toxicity of thuringiensin is much less than that of most chemical insecticides. Therefore, it shows great potential to become a very useful insecticide for controlling a wide range of insects.
Recent studies have shown that the production of thuringiensin could be improved by a net-draft-tube modified air-lift reactor (Tzeng, Y. M. and Young, Y. H., (1996) World J. Microbiol. Biotechnol. 12:32-37). During the period of fermentation, penicillin-G may enhance the production (Tzeng, Y. M. and Young, Y. H., (1995) Biotechnol. Prog. 11:231-234). These findings make thuringiensin more practical in terms of mass production. However, a lack of low-cost method for recovery of thuringiensin from fermentation broth has been the major rate-limiting step for industrial application. Traditional membrane ultrafiltration is not only expensive, but also time consuming and inefficient. Tzeng et al. (1999, Biotechnol. Prog. 15:580-586) developed a micellar-enhanced ultrafiltration method to facilitate the efficiency of recovery by using a surfactant and cetylpyridinium chloride (CPC). However, some drawbacks in this method include limited adsorption rate, toxicity of CPC, and micellar complex accumulation within the filter, thereby deteriorating the efficiency of ultrafiltration.